Diagnostic value of thyroid imaging reporting and data system combined with BRAFV600E mutation analysis in Bethesda categories III–V thyroid nodules

Fine needle aspiration biopsy is a crucial method for preoperative diagnosis of thyroid nodules. However, thyroid nodules classified as Bethesda categories III–V cannot obtain definite cytological results. Our aim was to study the diagnostic value of thyroid imaging reporting and data system combined with BRAFV600E mutation analysis in Bethesda categories III–V thyroid nodules, so as to provide more precise direction for the follow-up treatments. A total of 174 Bethesda categories III–V thyroid nodules performed TIRADS and BRAFV600E mutation analysis were included in the study. We retrospectively analyzed the ultrasound features as well as the results of BRAFV600E mutation of the 174 thyroid nodules. In the multiple regression analysis models, ultrasound features including lobulated or irregular margin, punctate echogenic foci, and shape with taller-than-wide were statistically significant in malignant nodules (p < 0.05). The area under the curve of the combination of TIRADS and BRAFV600E increased to 0.925, which were much higher than TIRADS (0.861) and BRAFV600E (0.804) separately. Combined diagnosis was of the greatest value to identify Bethesda III–V thyroid nodules definitely, especially with higher sensitivity (93%) and accuracy (90%).

Fine needle aspiration (FNA) biopsy of the thyroid has been adopted as a fundamental procedure for diagnosing suspicious nodules indicated by Kwak's Thyroid Imaging Reporting and Data System (TIRADS) 1 . Bethesda System 2 recommended by the American thyroid association (ATA) 3 to report the results of FNA. The Bethesda System showed a relatively high accuracy in reporting FNA cytology, with 89% of samples being satisfactory for interpretation, 74% reported as definitively benign and 5% as definitively malignant 4 . However, approximately 20-30% of the cytology samples still belong to indeterminate diagnostic results 5 , that is, Bethesda categories III-V (III: unclear cellular atypical lesion or unclear follicular lesion AUS/FLUS; IV: follicular lesion or suspicious follicular tumor SFN/FN; V: suspicious malignant tumor SM), with a certain degree of malignancy. Then, in order to obtain a definitive diagnosis, surgery is selected by a number of patients with nodules in these cytological categories 6 . Due to the aggressively biopsying small nodules and performing extensive surgeries, the detection rate of thyroid cancer has increased dramatically in recent decades 7 . However, the overall mortality rate of thyroid cancer is not significant 7 , and thyroid cancer remains one of the least deadly human cancers. With the increasing cost of surgery, which proves 4.7-6.5 times more expensive than active surveillance 8 , the excessive medical treatments in thyroid nodules are brought into focus. Therefore, it is imperative to diagnose malignant lesions among indeterminate thyroid nodules precisely to avoid unnecessary operations and achieve a good cost effectiveness.
To fulfill this goal, molecular marker profiling is performed as an auxiliary diagnosis of thyroid nodules. BRAF is a member of the raf family of serine/threonine protein kinases, which has been shown to be mutated in 7% of all cancers, promoting to the activation of downstream transcription factors, as well as leading to cell differentiation, proliferation, growth, and apoptosis 9 . Additionally, it is suggested that BRAF V600E mutation is associated with poor prognosis of thyroid cancer 10 [11][12][13][14] . In a word, the utility of BRAF V600E analysis for thyroid nodules enhances the diagnostic value of FNA cytology. However, in the diagnosis of indeterminate nodules, a previous study found that BRAF V600E mutation seemed to play a limited role owing to its low sensitivity 15 .
To date, the value of TIRADS combined with BRAF V600E mutation in distinguishing malignant from benign lesions in indeterminate thyroid nodules remains controversial. Wu et al. 6 have demonstrated that BRAF V600E mutation analysis and TIRADS classification were reliable diagnostic tools in Bethesda categories I(undiagnosed or unsatisfactory specimen ND/UNS), III, and V thyroid nodules in a Chinese population. Conversely, Bethesda category IV thyroid nodule was rarely associated with BRAF V600E mutation 16 . Thus, this study aimed to evaluate the diagnostic values of TIRADS and BRAF V600E mutation analysis in Bethesda categories III-V thyroid nodules, so as to provide clinical decision-making information for patients with thyroid nodules who failed to obtain a definite diagnosis after FNA.

Results
Clinical and ultrasonic features. A total of 172 patients including 42 males and 130 females with 174 indeterminate thyroid nodules (Bethesda categories III-V) were included in our research. The clinical and ultrasonic features are displayed in Table 1.
The mean size of malignant nodules were 12 ± 7 mm, which displayed significantly smaller maximal diameters than benign ones (p < 0.001). In univariate analysis, shape, echogenicity, margin, echogenic foci, acoustic halo, and blood flow proved statistical significance between benign and malignant nodules (p < 0.05). In multiple www.nature.com/scientificreports/ regression analysis, shape with taller-than-wide, punctate echogenic foci, lobulated or irregular margin were ultrasonography features showing significant statistically (p < 0.05).

The diagnostic efficacy of TIRADS, BRAF V600E mutation analysis and combined diagnosis in
Bethesda categories III-V thyroid nodules.. The specific information of thyroid nodules from different diagnostic methods is presented in

Discussion
It was reported that the histopathology malignancy rates was 98% for cytology malignant cases (Bethesda category VI) through FNA 17 . Cha et al. found the malignant rates in surgical cases are as follows for Bethesda category: III (50.6%), IV (52.3%), and V (90.7%) 18 , and meanwhile our study indicated that the malignant risks of III, IV(follicular adenoma confirmed by the postoperative pathology) and V were 34.6%, 0% and 80.2%, respectively, which were higher than the estimated malignant risks of the Bethesda system in III and V nodules 2 , indicating the conservative approaches of pathologists. These three categories of Bethesda system are full of uncertainty and are managed entirely differently by a number of centers. Therefore, appropriate diagnostic methods were required to distinguish malignant and benign lesions among these nodules. The present study discovered that the combination of TIRADS and BRAF V600E mutation analysis achieved a better diagnostic efficiency in differentiating Bethesda categories III-V thyroid nodules. A study indicated that the incidence rate of thyroid cancer has increased significantly among the population aged 15-39, especially in women 19 . In our research, malignant thyroid nodules has a higher incidence in women than men and the average age of patients subjected to thyroid cancer was 44, which is consistent with Lim's finding 20 . Thereby, females with questionable thyroid nodule are supposed to be treated more carefully.
Ultrasound is the primary and preferred examination method for risk stratification of thyroid nodules, which could guide the follow-up measures. We figured out that the maximum diameter of a benign nodule were larger than a malignant one (p < 0.001), cohering with the Kwak's study 21 . In our multiple logistic regression analysis, shape with taller than wide, lobulated or irregular margin, and punctate echogenic foci were independent risk factors (p < 0.05), consistent with previous studies [22][23][24] . It was certain that the above-mentioned ultrasound signs could play a vital role in diagnosing indeterminate thyroid nodules. In current study, the ROC curve showed that the best cut-off for TIRADS classification was 4b, consistent with findings perceived by Zhang et al. 25,26 . Nevertheless, our research suggested a slightly higher sensitivity of TIRADS compared to the Zhang's discovery which included Bethesda categories I-VI (85% versus 73.1%) and a higher PPV compared to a finding by Singaporewalla et al. 27 (81.7% versus 60%). According to the guidelines, the risks of malignancy of nodules classified as TIRADS 3, 4a, 4b, 4c, and 5 were < 2%, 2-10%, 10-50%, 50-95%, and ≥ 95%, respectively 1 , consistent with our findings in part. The malignant rates of TIRADS 4a, and 4b were 25.4% (15/59), and 66.7% (32/48) in our study, which were higher than the guideline 1 . This was probably on account of the cautious assessments of sonographers and differences in populations. At the same time, our data demonstrated that 95.4% (166/174) thyroid nodules belonged to solid nodules, which had no statistically significance in the differential diagnosis of benign and malignant www.nature.com/scientificreports/ nodules. In practice, a solid thyroid nodule without other suspicious ultrasonic characteristics was inclined to a benign one classified as TIRADS 3 in our institution. Even though there were limitations in TIRADS, it still provided a standardized risk stratification for thyroid nodules to guide follow-up managements for clinicians. The prevalence of BRAF mutation in thyroid cancer was 77% (77/100), in our series which was comparable to another report from Kim et al. 28 . A correlation between the BRAF V600E mutation and aggressive disease features, including lymph node metastases, invasion, and recurrence, has been reported by Martina et al. 29 . BRAF analysis boosted the accuracy of cytology and possessed a particular value for indeterminate nodules in the Chinese population 30 . In our study, the malignant rate of nodules with BRAF V600E mutation was 86.5% (77/89) in Bethesda categories III-V, consistent with findings by Zhu et al. 31,32 . Seo et al. 33 found that BRAF mutation analysis showed additional diagnostic value in thyroid nodules with Bethesda category V alone even when the nodules do not show suspicious ultrasonic features. A meta-analysis of 32 studies found that the overall specificity for BRAF V600E in the diagnosis of thyroid cancers was 100% in indeterminate nodules 15 , conforming to a finding by Han et al. 34 . In general, BRAF V600E gene had a significant difference in Bethesda categories III-V thyroid nodules. If the indeterminate thyroid nodule with a mutant BRAF V600E gene, repeat FNA might be considered to supplement malignancy risk assessment 3 .
The diagnostic sensitivity, accuracy, and AUC of the combined diagnosis were much higher than those of TIRADS or BRAF V600E separately, in accordance with Wu's finding 6 , improving the ability to diagnose malignant nodules and reduce false negatives. The malignant rate of thyroid nodules with BRAF V600E mutation and TIRADS classification 4b-5 was up to 89.4% (93/104) in our study, similar to the malignant rate of Bethesda category www.nature.com/scientificreports/ VI thyroid nodules 35 . Accordingly, TIRADS combined with BRAF V600E mutation analysis reached a valuable diagnostic efficacy for Bethesda III-V thyroid nodules. There were several limitations in the our study. Firstly, this study was a retrospective study. Patients with nodules of Bethesda categories III-IV mostly chose follow-up surveillance, and meanwhile, a part of patients with Bethesda categories V nodules turned to surgical treatment because of factors such as large nodules, mental tension, and mutant BRAF V600E gene, resulting in a certain degree of selection bias. Secondly, the single center with a small sample, which is still needed to be further verified by expanding the volume of sample and cooperating with other institutions. Lastly, we did not explore the predictive value of TIRADS and BRAF V600E in the staging and prognostic of thyroid carcinomas, which has been confirmed in another study 29 .
In summary, the combination of TIRADS and BRAF V600E had the highest diagnostic efficacy in Bethesda categories III-V thyroid nodules. For Bethesda categories III-V thyroid nodules with BRAF V600E mutation and TIRADS classification 4b-5, surgery should be recommended. Otherwise, regular follow-up ultrasound or repeat FNA were deemed to be appropriate.

Methods
Patients. This study enrolled a total of 172 patients aged from 17 to 76 with 174 indeterminate thyroid nodules who underwent FNA, two dimension (2D) ultrasonography and BRAF V600E mutation analysis in the First Affiliated Hospital of Nanjing Medical University from February 2018 to November 2021. Inclusion crite- This retrospective study was approved by the Institutional Ethical Committee in the First Affiliated Hospital of Nanjing Medical University and written informed consent was obtained from all the patients before performing FNA and BRAF V600E analysis. All methods were conducted in accordance with the relevant guidelines and regulations.

Data availability
The datasets generated and analysed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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